Archer Daniels Midland Company

Andriy Sibirny

Charles Abbas

Andriy Voronovsky

Olena Ishchuk

Kostyantyn Dmytruk

Marta Semkiv

Barbara Kshanovska

Andriy Y Voronovsky

Oleh V Stasyk

Olena B Ryabova

Charles A Abbas

Olena Dmytruk

Marta V Semkiv

Kostyantyn V Dmytruk

Lidiia R Murashchenko

Methods of identifying genes conferring ethanol tolerance in yeasts, genes that confer ethanol tolerance, and mutant strains used to identify such genes are described. A gene herein designated HpETT1 was isolated from the yeast. Expression of HpETT1 in an ethanol sensitive mutantstrain designated 7E complimented ethanol sensitivity of the mutant. When multiple copies of the HpETT1 were integrated into the genome and overexpressed, the transformed strain demonstrated approximately 10-fold greater resistance to ethanol and resistance to the protein misfolding agent AZC. Expression of HpETT1 also increased ethanol tolerance in. HpEtt1 has 39% sequence identity to a previously identified protein fromdenoted MPE1, however, the MPE1 gene does not confer ethanol resistance to the 7E mutant. Another gene from the yeastwas identified that encodes an orthologue protein having 37% identity to HpETT1 herein designated PsETT1 and also confers ethanol resistance to the 7E mutant.

Genes conferring tolerance to ethanol and high temperature for yeasts

Described herein are new approaches for the selection ofstrains with increased ethanol production from hydrolyzed starch derived sugars. An industrial production strain ofAS400 was subjected to positive selection of mutants resistant to toxic concentrations of oxythiamine, trehalose, 3-bromopyruvate, glyoxylic acid, and glucosamine. The selected mutants are characterized by 5-8% increase in ethanol yield (g gof consumed glucose) as compared to the parental industrial ethanol-producing strain. A multiple-step selection approach that consisted of the sequential selection using glyoxylic acid, glucosamine and bromopyruvate as selective agents resulted in a 12% increase in ethanol yield during fermentation on industrial media. These results indicate that the selection methods provided herein are useful for producing a variety of strains that are promising candidates for industrial ethanol production.

Methods for the positive selection of ethanol overproducing mutants from

Described herein is a method to increase ethanol yield during alcoholic fermentation by expression a truncated versions of thePHO8 gene coding for vacuolar or cytosolic form of alkaline phosphatase, which expression lowers biomass accumulation while diverting greater carbon to ethanol production. Also described are as nucleic acid sequences and vectors for expression of the PHO8 gene and strains carrying the same. Strains containing intact PHO8 gene encoding vacuolar form of alkaline phosphatase, had slightly lower intracellular ATP levels with insignificant changes in biomass accumulation and up to 13% increase in ethanol production.

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Genes SWA2 and GAM1 from the yeast,, encoding &#x3b1;-amylase and glucoamylase, respectively, were cloned and expressed in. The expression was achieved by integration of the SWA2 and GAM1 genes into the chromosome ofunder operably linked to a strong constitutive promoter of the-glyceraldehyde-3-phosphate dehydrogenase gene (HpGAP. Resulting transformants acquired the ability to grow on a minimal medium containing soluble starch as a sole carbon source and can produce Ethanol at high-temperature fermentation from starch up to 10 g/L. A XYN2 gene encoding endoxylanase was obtained from the fungus, and a xlnD gene coding for &#x3b2;-xylosidase was obtained from the fungus. Co-expression of these genes was also achieved by integration into thechromosome under control of the HpGAP promoter. The resulting transformants were capable of growth on a minimal medium supplemented with birchwood xylan as a sole carbon source. Successful expression of xylanolytic enzymes resulted in a recipient strain capable of fermentation of birchwood xylan to ethanol at 48&#xb0; C. Further with co expression of the forgoing genes in astrain that overexpresses a pyruvate decarboxylase gene further improved ethanol production.

Recombinant genetic constructs and strains ofhaving significantly increased ethanol productivity with a simultaneous decreased production of xylitol during high-temperature xylose fermentation are disclosed. The constructs include aXYL1 gene encoding xylose reductase mutated to decrease affinity of the enzyme toward NADPH. The modified version of XYL1 gene under control of a strong constitutive HpGAP promoter was overexpressed in a &#x394;xyl1 background. A recombinantstrain overexpressing the mutated enzyme together with native xylitol dehydrogenase and xylulokinase in the &#x394;xyl1 background was also constructed. Xylose consumption, ethanol and xylitol production by the constructed strain were evaluated during high-temperature xylose fermentation (48&#xb0; C.). A significant increase in ethanol productivity (up to 7.4 times) was shown in the recombinant strain as compared with the wild type strain. Moreover, the xylitol production by the recombinant strain was reduced considerably: 0.9 mg&#x3c7; (L&#x3c7;h)versus 4.2 mg&#x3c7; (Lxh)by the wild type strain.

Methods and compositions for the production of ethanol from lignocellulosic starting materials are provided herein. Embodiments provide yeast cells of the genuswith one or more modifications, including, for example, an inactive acid trehalase gene, overexpression of xylulokinase, and/or overexpression of heat-shock protein 104.

A new approach for increase of ethanol yield during alcoholic fermentation via decrease of biomass accumulation by using ATP degrading enzymes is described. The part of theSSB1 gene coding for cytosolic ATPase domain of ribosome associated chaperon cloned into expression cassette under control of the glycerol-3-phosphate dehydrogenase gene (GPD1) promoter was introduced into theBY4742 strain. The recombinant strains were tested for their ability to grow and produce ethanol during glucose anaerobic and aerobic cultivations. Strains overexpressing ATPase domain of SSB1 possessed decreased concentration of intracellular ATP. They accumulated elevated amounts of ethanol and were characterized by decreased biomass accumulation as compared to the wild-type strain under both anaerobic and aerobic conditions. Similarly, the apyrase gene apy fromencoding ATP/ADP hydrolyzing phosphatase and ATPase domain of SSB1 gene ofwere co-expressed under the control of galactose-inducible GAL1 promoter. The recombinantstrains revealed slight reduction of biomass accumulation, while specific ATPase activity, ethanol accumulation and yield during alcoholic galactose fermentation under semi anaerobic conditions were increased.

Methods and compositions for the production of ethanol from lignocellulosic starting materials are provided herein. Embodiments of the invention provide methods of manipulating the carbon flux of a host cell transformed with plasmids of the invention. Plasmids of the invention may include nucleotides that encode pyruvate decarboxylase. In one embodiment, a strain of the thermotolerant yeastthat has been transformed with plasmids and polynucleotides of the invention is provided.

Growing community of inventors

Lviv, Ukraine

Andriy Sibirny